Cr(VI) is a known human carcinogen. After in vivo exposure to Cr(VI), DNA-Cr(III)-protein crosslinks are formed. Their persistence suggests they might be important to cancer initiation. It would be desirable to develop methods to identify and quantitate these crosslinks. In order to do so, it is necessary to to identify the nucleotide(s) and the amino acid(s) that are actually complexed to the chromium(III) and chemically synthesize the complexes for comparison. A panel of monoclonal antibodies could be raised against the synthetic crosslinks; these could be used in immunoassays to quantitate the crosslinks. This project proposes to answer many of the questions involved in developing such an assay. It proposes to use chemical synthesis to make Cr(III) complexes with the nucleotides (four) and amino acids (three) that are most likely to form these complexes and then produce polyclonal antibodies to these 12 species. Oligonucleotide-Cr(III)-peptide complexes will be synthesized and digested with proteases and DNases to determine how far the enzymes will digest with the chromium present. The polyclonal antibodies will be used to determine if they can detect the macromolecular crosslinks formed in vivo or the smaller species resulting from protease plus nuclease digestion, and if the immunoassays based on the antibodies can achieve the femtomole detection limits needed. Restriction digests with GC or AT specific endonucleases will be performed on DNA that has been crosslinked through Cr(III) to proteins in vitro, in bacteria and in animals, to determine if crosslink formation shows any preference for GC or AT regions. Chemical reduction procedures will be developed for in vitro experiments. The results of these studies will tell us whether footprinting studies would yield more information on DNA sequences specificity in crosslinking.